Flow-cytometry methods to investigate anti-platelet immunity Stephan Meinke, PhD Cell and Gene Therapy Group (Evren Alici) & Petter Höglund Research Group Center for Hematology and Regenerative Medicine (HERM), Department of Medicine Huddinge SFFF 2018, Karlskrona
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Flow-cytometry methods to investigate anti-platelet immunity
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Flow-cytometry methods to investigate anti-platelet
immunity
Stephan Meinke, PhDCell and Gene Therapy Group (Evren Alici) & Petter Höglund Research GroupCenter for Hematology and Regenerative Medicine (HERM), Department of Medicine Huddinge
How do we measure the effect ofplatelet transfusions?
Bleedings stop. Platelet counts in the blood increase.
Standardized measurement is the Corrected Count Increment (CCI)calculated from: Platelet increment (PI): difference in platelet count before and after
transfusion Body surface area (BSA) Platelet dose (PD)
CCI = PI x BSA x PD-1
A CCI of ≥7.5 x 109 m2/L is considered an acceptable response.
Stephan Meinke 3rd September 2018
Platelet transfusion refractoriness
The repeated failure to raise the platelet count by transfusion of standard platelets
One hour CCI < 5 x 109 m2/L in two consecutive transfusions
Stephan Meinke 3rd September 2018
Factors that affect the responseto platelet transfusions
The patient’s primary illness
Splenomegaly, leukemia, lymphoma, kidney failure
The patient’s current clinical status
Sepsis, bleeding, fever
The patient’s current treatment
Antibiotics, cytostatic drugs
The platelet unit’s storage time
Anti-platelet antibodies About one third of refractoriness cases have an immune component.
Legler et al., Ann Hematol, 74:185, 1997Doughty et al., Vox Sang, 66:200, 1994
Stephan Meinke 3rd September 2018
Immunity against allogeneic platelets
Platelets carry ABO blood group antigens.
There are 28 bi-allelic platelet-specific antigens (human platelet antigens, HPA) Antibodies against HPA-1a or HPA-5b alleles are most common in
cases of FNAIT.
Platelets express HLA class I antigens HLA-A, -B and -C Antibodies against HLA-A or -B alleles are the most common cause
for immune-mediated platelet transfusion failure.
Stephan Meinke 3rd September 2018
Antibody-mediated platelet destruction
Phagocytosis by cells expressing Fc receptors
Lysis by Natural Killer cells (?)
Complement-mediated lysis
Υ
Υ
Υ
ΥΥ
Υ
Υ
Υ
ΥΥ
NK
NK
v
Υ
Stephan Meinke 3rd September 2018
How many HLA-typed donors are neededto provide HLA-matched platelets to all patients?
Takahashi et al., Transfusion, 27:394, 1987 5 completely matched donors for 80 % of all patients
Japan: 5000, Europe: 18000, North America: 21000
Bolgiano et al., Transfusion, 29:306, 1989 Covering the transfusion needs for most patients (completely matched or one cross-
reacting allele) 1000-3000
Bub et al., Brazil J Hematol Hemother, 38:1, 2016 5 completely matched donors, or have one or two cross-reacting alleles
Complete match: 32000, 1x: 1710, 2x: 321
Stanworth et al., Br J Hematol, 171:297, 2015 Number of donors to provide 70% of patients with platelets that are either completely
matched or have one or two cross-reacting alleles 12000
Stephan Meinke 3rd September 2018
Acidic elution of HLA-moleculesto create universal donor platelets
Sugawara S, Abo T, Kumagai K.:
A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3.
J Immunol Methods. 1987 Jun 26;100(1-2):83-90.
Kurata Y, Oshida M, Take H, Furubayashi T, Nakao H, Tomiyama Y, Kanayama Y, Nagao N, Okubo Y, Yonezawa T, et al.:
New approach to eliminate HLA class I antigens from platelet surface without cell damage: acid treatment at pH 3.0.
Vox Sang. 1989;57(3):199-204.From: Achour,A., et al. Immunity 1998, 9, 199-208
Stephan Meinke 3rd September 2018
Clinical advantages of acid-treated platelets
They could survive and function in the circulation of patients with anti-HLA antibodies.
They can be produced from random donor platelets and would be available in acute situations.
They could be used as a complement to matched donor platelets if too few or no matching donors are available.
They could be used in hospitals that lack the resources for organizing a sufficient pool of matched donors.
Stephan Meinke 3rd September 2018
Transfusion of acid-treated platelets
From: Novotný VM, Doxiadis II, Brand A.: The reduction of HLA class I expression on platelets: a potential approach in themanagement of HLA-alloimmunized refractory patients. Transfus Med Rev. 1999 Apr;13(2):95-105.
!
Stephan Meinke 3rd September 2018
Why do we try to revive this approach?
The potential clinical advantages are big. No controlled clinical study has been performed so far.
In the case reports, different protocols for the acid-treatment were used.
Not all of them report, what quality controls they applied and whether they assessed the extent of HLA removal.
The status of the patients receiving the platelets differed strongly between the cases.
There were no studies that investigated if the acid treatment prevented anti-HLA antibody-mediated platelet destruction.
Stephan Meinke 3rd September 2018
HLA denaturation throughtreatment with low pH
HLA-A, B, C101 102 103 104 105
0
100
200
300
# C
ells
β2-microglobulin101 102 103 104 105
0
100
200
300
# C
ells
free HLA heavy chain101 102 103 104 105
0
100
200
300
# C
ells
isotype controls
untreated platelets
control-treated platelets
acid-treated platelets
Stephan Meinke 3rd September 2018
Platelet Immunofluorescence Test(PIFT)
Based on the method described by von dem Borne et al. 1978 Br J Haematol
donor platelets + patient serum with antibodies detection with anti-Fc antibodies
Calcein-labeled platelets were incubated for 10 min at 37°C in the presence of anti-HLA antiserum and active complement and then stained for C1q and C3c.
calcein
C1q
C3c
untreated control-treated acid-treated
0 102 103 104 105
0
102
103
104
105
14.3
0 102 103 104 105
0
102
103
104
105
16.9
0 102 103 104 105
0
102
103
104
105
15.5
0 102 103 104 105
0
102
103
104
105
17.3
0 102 103 104 105
0
102
103
104
105
4.04
0 102 103 104 105
0
102
103
104
105
4
Stephan Meinke 3rd September 2018
Phagocytosis assays withlabelled platelets
ΥΥ
Υ Υ
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ΥΥ
Υ
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ΥΥ
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Υ
Monocytes Platelets + patient serum Phagocytosis
Stephan Meinke 3rd September 2018
Phagocytosis assays withlabelled platelets
Platelets readily attach to monocytes.
So, the problem is to distinguish between platelets on the surface and inside the monocytes.
Stephan Meinke 3rd September 2018
Phagocytosis assays withlabelled platelets
Trypsin/EDTA
0 102 103 104 105
<FITC-A>: CFSE
0
103
104
105
<Pac
ific
Blue
-A>:
CD
42a
20.5 77.6
0.1581.76
0 102 103 104 105
<FITC-A>: CFSE
0
103
104
105
<Pac
ific
Blue
-A>:
CD
42a
47 23
2.1927.8
Stephan Meinke 3rd September 2018
Phagocytosis assays with pHrodo
0 102 103 104 1050
50
100
150
200
250
# C
ells
pHrodo
platelets in PBS
platelets at pH 2.9
pH in lysosomes: about 4.8
Stephan Meinke 3rd September 2018
Anti-HLA antibody-mediated phagocytosis
Platelets were incubated with a mouse monoclonal anti-HLA-A,B,C antibody washed and then added to PBMC at a 10:1 ratio. Gated on CD14+ monocytes.
0 102 103 104 1050
100
200
300
400
500
# C
ells 1.04
0 102 103 104 1050
100
200
300
400
500
# C
ells 1.14
0 102 103 104 1050
100
200
300
400
500
# C
ells 47.2
pHrodo
on ice 37°C + cytochalasin D 37°C
platelets without antibodies platelets incubated with antibodies
Platelets were incubated with a mouse monoclonal anti-HLA-A,B,C antibody washed and then added to PBMC at a 10:1 ratio. Gated on CD14+ monocytes.
platelets without antibodies platelets incubated with antibodies
Stephan Meinke 3rd September 2018
pHrodo bright events are gated based on the respective cytochalasin D-treated samples. Plotted are means and SD of six combinations of four different platelet concentrates and two donors in four experiments.